Many viruses, including herpes-, adeno-, and poxviruses, use powerful ATP-driven molecular motors to package their double stranded DNA genomes into viral capsid shells. The objective of this project is to understand the molecular-structural mechanism by which these motors operate, which is important because DNA packaging is a critical step in viral assembly and a potential target for antiviral drugs. An integrated experimental and computational approach will be used to study the bacteriophage T4 packaging motor, to date the only viral motor for which atomic structures are available and a defined in vitro packaging assay has been established. Single-molecule optical tweezers methods combined with site-directed mutagenesis and kinetic analyses will be used to test hypothesized roles of motor structural transitions in DNA packaging and link these transitions with individual kinetic steps in the ATP hydrolysis cycle. An integrated single-molecule fluorescence/optical tweezers approach incorporating labeled mutant and wild type subunits into the motor complex will be used to investigate how the multiple motor subunits coordinate their activities to rapidly package DNA. Molecular, structural, and kinetic modeling will be used to generate testable predictions regarding motor structure-function relationships to be probed by the experimental studies and to provide a detailed mechanistic interpretation of the experimental findings. The outcome of this project will be the first atomic-level model of a viral DNA packaging motor that links mechanical and chemical kinetics with structural transitions. The structural mechanisms revealed here, along with the advances in experimental and computational modeling techniques, will be widely applicable to other complex biomolecular machines.